CD1d-Dependent iNKT Cells Control DSS-Induced Colitis in a Mouse Model of IFNγ-Mediated Hyperinflammation by Increasing IL22-Secreting ILC3 Cells.

We have previously shown that CD1d-restricted iNKT cells suppress dysregulated IFNγ expression and intestinal inflammation in Yeti mice on the C57BL/6 background. Since type 3 innate lymphoid cells (ILC3s) in mesenteric lymph nodes (MLN) protect against intestinal inflammation in a CD1d-associated manner, we investigated whether crosstalk between iNKT cells and MLN ILC3s controls IFNγ-mediated intestinal inflammation in Yeti mice. We found that Yeti mice display increased levels of ILC3s and that iNKT cell deficiency in Yeti/CD1d KO mice decreases levels of IL22-producing ILC3s during DSS-induced colitis. This finding indicates that iNKT cells and ILC3s cooperate to regulate intestinal inflammation in Yeti mice. Yeti iNKT cells displayed a pronounced anti-inflammatory (IL4- or IL9-producing) phenotype during colitis. Their adoptive transfer to iNKT cell-deficient animals induced a significant increase in IL22 production by ILC3s, indicating that crosstalk between iNKT cells and ILC3s plays a critical role in modulating colitis in Yeti mice. Moreover, we showed that the IL9-producing subset of iNKT cells potently enhances IL22-producing ILC3s in vivo. Taken together, our results identify a central role of the iNKT cell-ILC3 axis in ameliorating IFNγ-mediated intestinal inflammation.

Hyperdynamic circulatory syndrome in a mouse model transgenic for SerpinB3.

INTRODUCTION AND OBJECTIVES: SerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivo and in vitro effects of SerpinB3 on systemic and splanchnic hemodynamics. MATERIAL AND METHODS: Different hemodynamic parameters were evaluated by ultrasonography in two colonies of mice (transgenic for human SerpinB3 and C57BL/6J controls) at baseline and after chronic carbon tetrachloride (CCl4) treatment. In vitro SerpinB3 effect on mesenteric microvessels of 5 Wistar-Kyoto rats was analyzed measuring its direct action on: (a) preconstricted arteries, (b) dose-response curves to phenylephrine, before and after inhibition of angiotensin II type 1 receptors with irbesartan. Hearts of SerpinB3 transgenic mice and of the corresponding controls were also analyzed by morphometric assessment. RESULTS: In SerpinB3 transgenic mice, cardiac output (51.6±21.5 vs 30.1±10.8ml/min, p=0.003), hepatic artery pulsatility index (0.85±0.13 vs 0.65±0.11, p<0.001) and portal vein blood flow (5.3±3.2 vs 3.1±1.8ml/min, p=0.03) were significantly increased, compared to controls. In vitro, recombinant SerpinB3 had no direct hemodynamic effect on mesenteric arteries, but it increased their sensitivity to phenylephrine-mediated vasoconstriction (p<0.01). This effect was suppressed by inhibiting angiotensin II type-1 receptors. CONCLUSIONS: In transgenic mice, SerpinB3 is associated with a hyperdynamic circulatory syndrome-like pattern, possibly mediated by angiotensin receptors.

Atrophy of mesenteric sympathetic innervation may contribute to splanchnic vasodilation in rat portal hypertension.

BACKGROUND AND AIMS: Portal hypertension is associated with downregulation of mRNA and proteins involved in adrenergic transmission in the superior mesenteric artery (SMA) in portal vein-ligated (PVL) and cirrhotic rats. We aimed to investigate whether SMA adrenergic dysfunction was accompanied by sympathetic nerve structural changes and whether it was extensive to resistance mesenteric arteries. We also attempted to localize the origin of mRNA of specific adrenergic genes. METHODS AND RESULTS: In situ hybridization showed tyrosine hydroxylase (Th) mRNA expression in neuronal bodies of superior mesenteric ganglia and inside axonal fibres surrounding proximal SMA sections. Comparison of SMA by Th immunohistochemistry, both in PVL and bile duct-ligated (BDL) rats, demonstrated a significant decrease in the number of nervous structures (69% PVL; 62% BDL), total nervous area (70% PVL; 52% BDL) and Th-stained nervous area (89% PVL; 64% BDL) compared with sham rats. A strong correlation was detected between the Th-stained nervous area and the haemodynamic parameters, mainly with SMA resistance (r=0.9, P<0.001 for PVL and r=0.75, P=0.018 for BDL). Western blot analysis of Th, dopamine beta-hydroxylase and synaptosome-associated protein of 25 kDa indicated a significant inhibition in protein expression (35-58%) in mesenteric resistance arteries from both portal hypertension models compared with sham. By contrast, nervous structure analysis and protein expression in renal arteries showed no differences between sham and PVL rats. CONCLUSION: Portal hypertension is associated with sympathetic nerve atrophy/regression in the mesenteric arterial vasculature that could contribute to the splanchnic vasodilation associated with portal hypertension.

JAK2V617F mutation in patients with splanchnic vein thrombosis.

BACKGROUND: Splanchnic vein thrombosis can be the presenting manifestation of myeloproliferative neoplasms. However, the diagnosis of a myeloproliferative neoplasm in these patients is often problematic, and more objective criteria are needed. AIM: To determine the frequency of the mutation JAK2V617F in patients with splanchnic vein thromboses. METHODS: A consecutive series of 108 adult patients with portal vein thrombosis (n = 77) and Budd-Chiari syndrome (n = 31) referred for hemostasis evaluation was retrospectively studied, with a median follow-up of 51 months (1-104). RESULTS: One or more prothrombotic risk factors were present in 63% of the patients. Twenty-four (22%) out of the 108 patients presented the JAK2V617F, including 2 cirrhotic patients. Most had a low mutated allele burden (median 16.5%). JAK2V617F was present in all four patients with a previous diagnosis of a myeloproliferative neoplasm. In nine JAK2V617F-positive patients, the diagnosis of a myeloproliferative neoplasm was made at the thrombosis work-up, during follow-up or after JAK2V617F detection. Among the other 11 patients carrying the mutation, 2 patients have died, 4 had no evidence suggesting a myeloproliferative neoplasm, 1 had a normal bone marrow biopsy, and the other 4 could not be persuaded to undergo a biopsy. Among the patients without an overt myeloproliferative neoplasm, 15 out of 99 (15%) presented the JAK2V617F mutation. None of the JAK2V617F-negative patients have developed signs of a myeloproliferative neoplasm during follow-up. CONCLUSIONS: Our findings suggest that JAK2V617F occurs in a high proportion of patients with splanchnic vein thrombosis, and reinforces the diagnostic utility of JAK2V617F testing in this setting.

The impact of JAK2 and MPL mutations on diagnosis and prognosis of splanchnic vein thrombosis: a report on 241 cases.

Myeloproliferative diseases (MPDs) represent the commonest cause of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT), but their diagnosis is hampered by changes secondary to portal hypertension, while their influence in the outcome of SVT remains unclear. We assessed the diagnostic and prognostic value of JAK2 and MPL515 mutations in 241 SVT patients (104 BCS, 137 PVT). JAK2V617F was found in 45% of BCS and 34% of PVT, while JAK2 exon 12 and MPL515 mutations were not detected. JAK2V617F was found in 96.5% of patients with bone marrow (BM) changes specific for MPD and endogenous erythoid colonies, but also in 58% of those with only one feature and in 7% of those with neither feature. Stratifying MPD diagnosis first on JAK2V617F detection would have avoided BM investigations in 40% of the patients. In BCS, presence of MPD carried significantly poorer baseline prognostic features, required hepatic decompression procedures earlier, but had no impact on 5-year survival. Our results suggest that JAK2V617F testing should replace BM investigations as initial test for MPD in patients with SVT. Underlying MPD is associated with severe forms of BCS, but current therapy appears to offset deleterious effects of MPD on the medium-term outcome.

Impact of genetic background and aging on mesenteric collateral growth capacity in Fischer 344, Brown Norway, and Fischer 344 x Brown Norway hybrid rats.

Available studies indicate that both genetic background and aging influence collateral growth capacity, but it is not known how their combination affects collateral growth. We evaluated collateral growth induced by ileal artery ligation in Fischer 344 (F344), Brown Norway (BN), and the first generation hybrid of F344 x BN (F1) rats available for aging research from the National Institute on Aging. Collateral growth was determined by paired diameter measurements in anesthetized rats immediately and 7 days postligation. In 3-mo-old rats, significant collateral growth occurred only in BN (35% +/- 11%, P < 0.001). The endothelial cell number in arterial cross sections was also determined, since this precedes shear-mediated luminal expansion. When compared with the same animal controls, the intimal cell number was increased only in BN rats (92% +/- 21%, P < 0.001). The increase in intimal cell number and the degree of collateral luminal expansion in BN rats was not affected by age from 3 to 24 mo. Immunohistochemical studies demonstrated that intimal cell proliferation was much greater in the collaterals of BN than of F1 rats. The remarkable difference between these three strains of rats used in aging research and the lack of an age-related impairment in the BN rats are novel observations. These rat strains mimic clinical observations of interindividual variation in collateral growth capacity and the impact of age on arteriogenesis and should be useful models to investigate the molecular mechanisms responsible for such differences.

Characterization of the non-adrenergic/non-cholinergic response to perivascular nerve stimulation in the double-perfused mesenteric bed of the mouse.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP), a capsaicin-sensitive neuromodulator of splanchnic vascular tone in several animal species, remains poorly investigated in mouse models. We therefore assessed whether endogenous CGRP is a non-adrenergic/non-cholinergic (NANC) neuromodulator in the mesenteric vascular bed of the mouse. EXPERIMENTAL APPROACH: Arterial and venous changes in perfusion pressure in response to perivascular nerve stimulation (PNS) were monitored in the mouse mesenteric bed under basal conditions or precontracted with KCl (artery) or U46619 (vein) in circuits pretreated with guanethidine, atropine, indomethacin and prazosin. Arterial responses to NANC were also characterized with a CGRP1 antagonist, halphaCGRP8-37. Finally, the PNS-induced release of arterial CGRP was measured by enzyme immunoassay. KEY RESULTS: HalphaCGRP8-37 enhanced PNS-induced arterial increases in perfusion pressure under basal tone. PNS-induced stimulation of NANC triggered an halphaCGRP8-37 or capsaicin- sensitive reduction in perfusion pressure of the pre-contracted arterial bed only. Chemical removal of the endothelium inhibited PNS- and halphaCGRP- induced reduction in perfusion pressure in the arterial mesenteric bed. Responses to NANC nerves were reduced by guanylate and adenylate cyclase inhibitors (1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (ODQ)) and [9-(tetrahydro-2-furanyl)-9H-purin-6-amine] (SQ 22,536), respectively. A neuronal NOS inhibitor (7-nitroindazole; 7-NI) also enhanced the response to NANC in vessels from wild-type, eNOS KO but not iNOS KO mice. Finally, PNS enhanced the release of immunoreactive CGRP from the perfused arterial mesenteric bed. CONCLUSIONS AND IMPLICATIONS: Our study demonstrates a role for CGRP in the NANC-dependent reduction in perfusion pressure of the arterial but not venous mesenteric bed of the mouse.

Preferential activity of Tie2 promoter in arteriolar endothelium.

The tyrosine kinase Tie2/Tek (the receptor for angiopoietins) is considered one of the most reliable markers of the endothelial phenotype, across organisms, organs, and developmental stages. However, endothelium is intrinsically heterogeneous in origin, composition and function, presenting an arteriolar/venular asymmetry. In this regard, the expression of Tie2 along the vascular tree, although thought to be homogenous, has not been systematically investigated. Therefore we questioned whether the activity of Tie2 promoter is uniform in the microvascular endothelium. To this end, we analyzed in situ the expression of the markers beta-galactosidase [LacZ(Tie2)] and green fluorescent protein (GFP) [GFP(Tie2)], placed under the Tie2 promoter in transgenic mice, in whole mount tissue samples, which allow the simultaneous evaluation of its relative distribution in various microvascular compartments. In the mesenteries of LacZ(Tie2) and GFP(Tie2) mice, we found that the activity of Tie2 promoter is asymmetrically distributed, being much stronger in arteries and arterioles than on the venular side of the vascular tree. This observation was replicated in the diaphragm of LacZ(Tie2) mice. The capillaries presented a mosaic pattern of Tie2 promoter activity. Stimulation of angiogenesis either by wounding, or by intraperitoneal injection of Vascular Endothelial Growth Factor (VEGF), revealed that the arteriolar/venular asymmetry is established at endothelial cellular level early during new capillary formation, even before the starting of the microvascular blood flow. In conclusion, a strong Tie2 promoter activity qualifies as a novel marker of the arteriolar phenotype in microvascular endothelium.

Cardiac neural crest ablation alters Id2 gene expression in the developing heart.

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.

Artemin is a vascular-derived neurotropic factor for developing sympathetic neurons.

Artemin (ARTN) is a member of the GDNF family of ligands and signals through the Ret/GFRalpha3 receptor complex. Characterization of ARTN- and GFRalpha3-deficient mice revealed similar abnormalities in the migration and axonal projection pattern of the entire sympathetic nervous system. This resulted in abnormal innervation of target tissues and consequent cell death due to deficiencies of target-derived neurotrophic support. ARTN is expressed along blood vessels and in cells nearby to sympathetic axonal projections. In the developing vasculature, ARTN is expressed in smooth muscle cells of the vessels, and it acts as a guidance factor that encourages sympathetic fibers to follow blood vessels as they project toward their final target tissues. The chemoattractive properties of ARTN were confirmed by the demonstration that sympathetic neuroblasts migrate and project axons toward ARTN-soaked beads implanted into mouse embryos.